Cellular internalization of large hydrophobic therapeutic agents such as proteins or nucleic acids is still a challenging task because of the presence of a plasma membrane, which constitutes an impermeable barrier for these molecules. In order to circumvent this problem, several methods of carrier-mediated delivery systems have been developed. Among these methods, much attention has been focused on the use of peptide-based delivery systems. The use of peptides with cell penetrating properties has many advantages because of various modifications that can be done to the peptide sequence. This allows the engineering of carriers addressing different cellular subdomains and/or transporting various types of cargoes.
Some cell penetrating peptides (CPPs) are designed from the sequences of membrane-interacting proteins, like fusion proteins, signal peptides, transmembrane domains, and antimicrobial peptides. These sequences, also called protein transduction domains (PTDs), prove to cross biological membranes without a carrier or a receptor, and to deliver peptides or proteins into intracellular compartments. Many studies suggest that the use of PTD-based peptides is of major significance for therapies against viral diseases or cancers (Victoria Del Gaizo, R. Molecular Therapy 2003, 7, 720-730; Harada, H., et al. Cancer Research 2002, 62, 2013). Among the studies, the penetratin from the Drosophila antennapedia homeodomain protein (Derossi, D., et al. Journal of Biological Chemistry 1994, 269, 10444), and Tat peptide derived from the HIV-1 genome have been used to improve the cellular uptake of peptides, proteins, or oligonucleotides.
Some CPPs, designated as amphipathic peptides, have been described. An amphipathic molecule can be divided into two regions: a hydrophilic (polar) region and a hydrophobic (non-polar) region. For peptides, their amphipathic properties arise either from primary structure or from secondary structure. Primary amphipathic peptides are defined as the sequential assembly of a domain of hydrophilic residues with a domain of hydrophobic residues. Secondary amphipathic peptides are generated by the conformational state which allows the positioning of hydrophobic residues and hydrophilic residues on opposite sides.
Other CPPs, such as polyarginine-based peptides, are synthesized and employed as a tool for intracellular delivery of therapeutics. The cationic guanidine moiety on the side chain of polyarginine-based peptides plays a critical role in cell penetrating properties (Futaki, S. International Journal of Pharmaceutics 2002, 245, 1-7; Futaki, S., et al. Journal of Biological Chemistry 2001, 276, 5836). Rothbard et al. have substituted arginine residues systemically with neutral alanine residues, which results in a significantly reduced cellular uptake (Vives, E., et al. Letters in Peptide Science 1997, 4, 429-436). They also have replaced other positively charged amino acids such as lysine, ornithine, histidine, and citrulline with arginine residues, and similar outcome is gained. This implies that arginine possesses unique cell penetrating properties when comparing with other positively charged amino acids.
Studies up to today reveal that currently used CPPs are cationic and limited because of toxicity and the lack of efficiency. As such, the disclosure is to provide a new family of CPPs which are anionic and exhibit low toxicity and high efficiency.